Project Summary/Abstract The ultimate objective of the proposed research is to enable the long-term banking of curative doses of human pancreatic islets for the reversal of type 1 diabetes and the amelioration of type 2 diabetes. Banking should reduce or prevent the deterioration of islets prior to use, facilitate tolerance induction, enable single rather than multiple islet infusions to achieve remission of type 1 diabetes, enable use of many more islets for therapeutic or research purposes, and have other advantages. We have already demonstrated, in unpublished results, that it is possible to vitrify and rewarm human pancreatic islets with excellent islet number recovery and excellent retention of viability (based on vital staining), glucose-stimulated insulin release (stimulation index), glucose-stimulated oxygen consumption, and ability to reverse induced diabetes in mice with minimal islet doses. The goal of the proposed research is to demonstrate that it is possible to scale up from the islet numbers vitrified to date (up to 13,000 IEQs) to curative doses of islets (450,000-720,000) with no significant changes in cryoprotection technique, cooling rate, or warming rate. This goal will be achieved by improving the spatial efficiency of the apparatus already created while simultaneously increasing the carrying capacity of that apparatus. Aim 1, milestone 1 is devoted to optimizing the balance between the rate of introduction of cryoprotective agents and the behavior of the islets within one specific and nominally optimal device design. Aim 1, milestone 2 will be devoted to exploring the design space of the device based on empirical observations of the responses of islets to different carrying capacity models. In Aim 2, we will fabricate and preliminarily test the optimized device resulting from the information gained in Aim 1. This device will be mostly automated to enable use by relatively unskilled personnel and to prevent variations caused by operator factors or operator error. In Aim 3, we will test the optimized device and method by actually vitrifying islets in densities within the device equivalent to the densities that will be required for preserving fully curative clinical islet doses, and the results will be evaluated independently by the City of Hope using the above- described endpoints. Finally, in Aim 4, we will obtain an objective evaluation of the barriers to clinical use of our methods and device as prepared by the City of Hope, and we will prepare a plan for overcoming any remaining barriers to clinical application based on the City of Hope report. This should enable us to move forward toward clinical trials of vitrified curative doses of human pancreatic islets.